A simple and reliable method is described here for the identification and relative quantification of proteins in complex mixtures using two-dimensional liquid chromatography/tandem mass spectrometry. The method is based on the classical proteomic analysis where proteins are digested with trypsin and the resulting peptides are separated by multidimensional liquid chromatography. The separated peptides are analyzed by tandem mass spectrometry and identified via a database search algorithm such as SEQUEST. The peak areas (integrated ion counts over the peptide elution time) of all identified peptides are calculated, and the relative concentration of each protein is determined by comparing the peak areas of all peptides from that protein in one sample versus those from the other. Using this strategy, we compared the relative level of protein expression of A431 cells (an epidermal cell line) grown in the presence or absence of epidermal growth factor (EGF). Our results are consistent with the published observations of the transient effects of EGF. In addition, the difference in the concentrations of several phosphopeptides determined in our studies suggests the possibility of several new targets involved in the EGF cell-signaling pathway. This global protein identification and quantification technology should prove to be a valuable means for comparing proteomes in biological samples subjected to differential treatments.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.

Methylation of lysine and arginine is known to be critical in cellular processes. However, methylation of other amino acidic residues has been largely overlooked. Here, we report a systematic screening for methylation of side chains of aspartate and glutamate (D/E-methylation), involving exhaustive nano-HPLC/MS/MS, a protein sequence database search, and manual verification. The putative D/E-methylated peptides were confirmed by MS/MS of synthetic peptides. Our analysis identified several D/E-methylation substrate proteins and their modification sites in human and yeast cells. To our knowledge, this is the first report conclusively identifying in vivo D/E-methylation substrates and their modification sites in eukaryotic cells, demonstrating that D/E-methylations are abundant protein modifications. The substrate proteins identified here provide a stepping stone for future biochemical characterization of protein methylation pathways.

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